DNA double-strand breaks (DSBs) are highly toxic DNA lesions that can occur spontaneously during DNA replication, but also can be introduced by exposure to ionizing radiation (IR) or to chemical mutagens. DSBs can lead to cell death, mutations and cancer, and defects in DSB repair (DSBR) underlie many human diseases, including disorders associated with cancer predisposition, radiosensitivity, immune dysfunction, neurodegeneration and premature aging. Nuclear Ubiquitous Casein and Cyclin-dependent Kinases Substrate (NUCKS) is a 27 kD, DNA-binding and vertebrate-specific protein with unknown function. NUCKS is abundant in rapidly growing cells and overexpressed in a variety of human cancers. Of particular interest, the NUCKS gene is located on human chromosome 1q32.1, a region that is commonly gained in breast cancer. Based on sequence homology to RAD51 Associated Protein 1 (RAD51AP1), a critical factor in homologous recombination repair (HRR), we have tested and determined a role for NUCKS in DSBR. Our results show that human cells with NUCKS knockdown are hypersensitive to IR and to chemical mutagens, and that NUCKS regulates not only HRR, but also cellular resistance to IR in G1-phase cells. In preliminary experiments using mice with heterozygous Trp53, we find that impaired Nucks function significantly increases the susceptibility to IR-induced tumors, alters the tumor spectrum and promotes metastasis. We now intend to further define how NUCKS regulates DSBR capacity using biochemical and cell-based assays (Aims 1 & 2). In addition, we will carry out a systematic study to further determine the phenotypic consequences of Nucks disruption in mice (Aim 2). Our findings showing a role for NUCKS in DSBR and in preventing radiation carcinogenesis are the first evidence for a biological function of NUCKS, a protein that was discovered more than 25 years ago. Given the importance of DSBR and HRR in tumor suppression and in the removal of DNA lesions induced by IR and other environmental mutagens, the results from our investigation will have direct relevance to risk predictions for health from environmental factors. In addition, our studies may also contribute to the improved diagnosis, prevention and treatment of cancer.